Germline Testing in a Cohort of Patients at High Risk of Hereditary Cancer Predisposition Syndromes: First Two-Year Results from South Italy

Germline pathogenic variants (PVs) in oncogenes and tumour suppressor genes are responsible for 5 to 10% of all diagnosed cancers, which are commonly known as hereditary cancer predisposition syndromes (HCPS). A total of 104 individuals at high risk of HCPS were selected by genetic counselling for genetic testing in the past 2 years. Most of them were subjects having a personal and family history of breast cancer (BC) selected according to current established criteria. Genes analysis involved in HCPS was assessed by next-generation sequencing (NGS) using a custom cancer panel with high- and moderate-risk susceptibility genes. Germline PVs were identified in 17 of 104 individuals (16.3%) analysed, while variants of uncertain significance (VUS) were identified in 21/104 (20.2%) cases. Concerning the germline PVs distribution among the 13 BC individuals with positive findings, 8/13 (61.5%) were in the BRCA1/2 genes, whereas 5/13 (38.4%) were in other high- or moderate-risk genes including PALB2, TP53, ATM and CHEK2. NGS genetic testing showed that 6/13 (46.1%) of the PVs observed in BC patients were detected in triple-negative BC. Interestingly, the likelihood of carrying the PVs in the moderate-to-high-risk genes calculated by the cancer risk model BOADICEA was significantly higher in pathogenic variant carriers than in negative subjects. Collectively, this study shows that multigene panel testing can offer an effective diagnostic approach for patients at high risk of hereditary cancers.


Introduction
Currently, inherited germline pathogenic variants in oncogenes and tumour suppressor genes are responsible for a small minority of cancers, around 5 to 10% of all diagnosed cancer cases, which are referred to as hereditary cancer predisposition syndrome (HCPS) [1]. More than 200 HCPS types and the associated genes have been described, which are normally driven by the presence of pathogenic variants in only one gene which confers an augmented risk of developing tumours at an early age in the affected individuals [2]. The majority of HCPS exhibit an autosomal dominant inheritance and include hereditary breast and ovarian cancer syndrome (HBOC), Lynch syndrome, Li-Fraumeni syndrome (LFS) and

Patients and Enrolling Criteria
A total of 104 patients were selected after genetic counselling at the Medical Genetics Unit (Mater Domini University Hospital at Catanzaro) between September 2019 and January 2022 for the NGS genetic test according to the family and personal criteria established by the National Comprehensive Cancer Network (NCCN) and the Associazione Italiana di oncologia Medica [7,8]. Genetic counselling was performed to evaluate the patient's cancer history (clinical diagnosis, age of first cancer, histological stage) and family history of cancer (number of affected relatives).
The inclusion criteria for the NGS genetic tests were: (1) women with BC and OC; males with BC; women with triple-negative breast cancer (TNBC) < 60 years; women with BC < 36 years; women with bilateral BC < 50 years; not mucinous and not borderline OC at any age; metastatic pancreatic adenocarcinoma; metastatic prostatic carcinoma.
(2) Personal history of breast cancer diagnosed < 50 years and at least one first-degree relative with nonmucinous and nonborderline OC at any age; BC < 50 years; male BC; bilateral BC; metastatic pancreatic adenocarcinoma and metastatic prostatic carcinoma.
In addition to the selection criteria for the enrolled patients described above, the cancer risk model called Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) was used for the HBOC and prostate cancer patients [10] to estimate the likelihood of carrying BRCA1, BRCA2, PALB2, CHEK2, ATM, BARD1, RAD51D, RAD51C and BRIP1 pathogenic variants (PVs). Other specific criteria for HPCS were used for patients with multiple cancer types, metastatic pancreatic adenocarcinoma diagnosed at any age and patients with pancreatic cancer or kidney cancer having a family history of cancer.

Genetic Testing: DNA Extraction and NGS
Genomic DNAs from patients were extracted from blood samples after signing informed consent forms using the NLM DNA extraction kit (Nuclear Laser Medicine) as previously described [11][12][13]. We designed two Ion Ampliseq On-Demand panels to explore, using NGS, the mutational status of the most frequently altered genes in HCPS. The panel includes BRCA1, BRCA2, ATM, PALB2, TP53, CHEK2, MLH1, MSH2, MSH6 and PMS2 (Thermo Fisher Scientific, Waltham, MA, USA) covering the full coding exons plus padding regions of the above-described genes. Libraries were constructed and purified on the Ion Chef Instrument according to the Ampliseq manufacturer's instructions. Subsequently, libraries were sequenced by the Ion GeneStudio S5 System (Thermo Fisher Scientific, Waltham, MA, USA).

Variant Analysis (Classification): Germline Calling Variants and Filtering
Only germline variants with an allele frequency < 0.01 based on allele frequencies found in GnomAD were retained for further investigation. Sequence variation databases such as ClinVar [14] and LOVD [15] were used to classify variants already reported, and when no data was available, the variant was classified following the American College of Medical Genetics (ACMG) criteria [16]. VUS variants were also classified using the semiquantitative, hierarchical evidence-based rules for the locus interpretation (Sherlock) method [17]. In addition, for VUS variants, computational prediction tools were used to predict the effect of the amino acid substitution on the protein function and structure. A total of 41/104 (39.4%) patients were selected for genetic testing following criteria 1 (Figure 2, see Materials and Methods for details), 24/104 (23.1%) patients were selected for genetic testing following criteria 2 and 17/104 (16.3%) following criteria 3. In addition, 17/104 (16.3%) patients did not meet the AIOM criteria but had personal and family history and were considered borderline, and 5/104 (4.8%) patients were selected using other criteria since they were not affected by tumours described in the AIOM criteria, such as uterine cancer, colon and LFS ( Figure 2). A total of 41/104 (39.4%) patients were selected for genetic testing following criteria 1 (Figure 2, see Materials and Methods for details), 24/104 (23.1%) patients were selected for genetic testing following criteria 2 and 17/104 (16.3%) following criteria 3. In addition, 17/104 (16.3%) patients did not meet the AIOM criteria but had personal and family history and were considered borderline, and 5/104 (4.8%) patients were selected using other criteria since they were not affected by tumours described in the AIOM criteria, such as uterine cancer, colon and LFS ( Figure 2). Women with BC and OC; males with BC; women with triple-negative breast cancer (TNBC) < 60 years; women with BC < 36 years; women with bilateral BC < 50 years; not mucinous and not borderline OC at any age; metastatic pancreatic adenocarcinoma; metastatic prostatic carcinoma. (2) Personal history of breast cancer diagnosed < 50 years and at least one first-degree relative with nonmucinous and nonborderline OC at any age; BC < 50 years; male BC; bilateral BC; metastatic pancreatic adenocarcinoma and metastatic prostatic carcinoma. (3) Personal history of BC > 50 years and family history of breast, ovarian cancer, metastatic prostatic carcinoma and metastatic pancreatic adenocarcinoma in 2 or more first-degree relatives (one of which in the first degree with the proband). (4) Presence of personal and family history that did not meet AIOM criteria. (5) Patients that were not affected by tumours described by AIOM criteria.
The study cohort included a total of 104 patients (94 females and 10 males; mean age of diagnosis 50.2, range 21-84 years, Figure 3A) with a clinical suspicion of hereditary cancer predisposition syndromes (HCPS) based on individual and family cancer history. A total of 26/104 (25%) patients were diagnosed with cancers before the age of 40 years, whereas 78/104 (75%) were diagnosed after 40 years of age ( Figure 3B). At the first diagnosis, eighty-eight had breast cancer (BC), five had ovarian cancer (OC), five had prostate cancer (PC), one had colon cancer, one had pancreatic cancer, one had LFS, one had kidney cancer and two had uterine cancer ( Figure 3C). Considering the 88 BC patients, 86 were monolateral and 2 were bilateral. Relating to BC histology, 72/88 (81.8%) had invasive ductal carcinoma (CDI), 4/88 (4.5%) had ductal carcinoma in situ (DCIS), 6/88 (6.8%) had invasive lobular carcinoma (CLI) and 6/88 (6.8%) had a rare BC histotype ( Figure 3D (1) Women with BC and OC; males with BC; women with triple-negative breast cancer (TNBC) < 60 years; women with BC < 36 years; women with bilateral BC < 50 years; not mucinous and not borderline OC at any age; metastatic pancreatic adenocarcinoma; metastatic prostatic carcinoma. (2) Personal history of breast cancer diagnosed < 50 years and at least one first-degree relative with nonmucinous and nonborderline OC at any age; BC < 50 years; male BC; bilateral BC; metastatic pancreatic adenocarcinoma and metastatic prostatic carcinoma. (3) Personal history of BC > 50 years and family history of breast, ovarian cancer, metastatic prostatic carcinoma and metastatic pancreatic adenocarcinoma in 2 or more first-degree relatives (one of which in the first degree with the proband). (4) Presence of personal and family history that did not meet AIOM criteria. (5) Patients that were not affected by tumours described by AIOM criteria.
The detailed information for each enrolled patient, including patient ID, age, sex, age of diagnosis, type of cancer, histological grade, molecular subtype, cancer onset, the occurrence of multiple tumours and the presence of affected first-and second-degree relatives, and BOADICEA scores are listed in Supplementary Table S1.   The detailed information for each enrolled patient, including patient ID, age, sex, age of diagnosis, type of cancer, histological grade, molecular subtype, cancer onset, the occurrence of multiple tumours and the presence of affected first-and second-degree relatives, and BOADICEA scores are listed in Supplementary Table S1.

Likelihood of Carrying PVs in the Moderate-to-High-Risk Genes Calculated by the Cancer Risk Model BOADICEA in HBOC and Prostate Cancer Patients
Currently, several cancer risk models are used to predict the risks of developing HBOC and prostate cancer and to calculate the likelihood of carrying PVs in the moderate-tohigh-risk genes. In this context, BOADICEA is considered the most accurate algorithm able to predict combined BRCA1/2 pathogenic variants with respect to other predictor models such as BRCAPRO, Penn II and Myriad [18]. In addition, the last V5 version of BOADICEA incorporates the effects of pathogenic variants (PVs), not only in BRCA1 and BRCA2 genes, but also in PALB2, CHEK2, ATM and BARD1 for the breast cancer model and RAD51D, RAD51C and BRIP1 for the ovarian cancer model [19]. The likelihood of carrying PVs for each patient is calculated by the BOADICEA model based on personal and family cancer history, mammographic density, histology, molecular subtype, hormonal risk factors and lifestyle.
Therefore, the BOADICEA prediction model was used to calculate the likelihood of carrying pathogenic variants in the moderate-to-high-risk genes in eighty-eight BC, five OC and three PC patients (n = 96) using a 10% pretest probability threshold. A total of 51/96 (53.1%) patients having BOADICEA > 10% probability were classified as high risk, whereas 45/96 (46.8%) patients with a percentage ≤ 10% were considered as low risk to be carriers of pathogenic variants in BRCA1, BRCA2, PALB2, CHEK2, ATM, BARD1, RAD51D, RAD51C and BRIP1 genes ( Figure 4A). For each patient, we also showed the risk in the well-known high-penetrance cancer risk alleles BRCA1 and BRCA2 with respect to other moderate-penetrance alleles including PALB2, CHEK2, ATM, BARD1, RAD51D, RAD51C and BRIP1 ( Figure 4B).

Genetic Testing and Variants Distribution
Gene panel sequencing yielded germline uncommon variants in 38 of 104 individuals (36.5%). No variants were detected in 66 individuals (63.4%), while 21 (20.2%) of the subjects had at least one VUS, and 17 (16.3%) had variants which were P/LP ( Figure 5A, B). Pathogenic variants (P) were detected in 13 patients (12.5%), whereas likely pathogenic variants (LP) were found in 4 (3.8%) of the subjects enrolled in this study.
Three of seventeen (17.6%) identified LP/P pathogenic variants were missense, four nonsense (23.5%) variants, three frameshift variants (17.6%) and seven splice-site variants (41.1%). The distribution of variants by effect is shown in Figure 5F. The complete list of pathogenic and likely pathogenic variants identified in this study, and the further details, including HGVS nomenclature, allelic frequencies, variant type, Clinvar classification and tumour type, is shown in Table 1.
Of the 17 P/LP variants detected by the NGS panel testing (Table 1), ten fulfilled AIOM criteria 1 (58.8%), four fulfilled criteria 3 (23.5%), one did not fulfil criteria (PALB2:c.1451T>A) and two patients were not affected by tumours described by AIOM, and thus were included considering other specific criteria, including one LFS and one colon cancer (11.7%, Figure 5C). In the colon cancer patient, we found the MSH2 PV (c.1204del), whereas TP53 PV (c.645delT) was found in a patient with LFS. A total of 82.3% of patients who received positive results in this study fulfilled the AIOM testing criteria. Thirteen pathogenic variants (PVs) (12.5%) were found in patients with BC, one patient

VUS, Variants Classification by ACMG Guidelines and Reclassification by SHERLOC Framework
In total, 24 VUS were found in 21 patients, since 2 of these patients (281/21 and 279/21) had more than one VUS. A detailed list of VUS variants identified in this study is provided in  Table  S1). Considering a distinction for pathology, among the sixteen BC patients with no other LP/P variants, three harboured VUS in the BRCA1 gene (18.7%), four patients harboured VUS in the ATM (25%), five VUS in CHEK2 (31.2%), one in MSH6 gene (6.2%), one in MLH1 (6.2%) and two in PMS2 (12.5%) ( Table 2). As regards the germline variant type, most were missense variants (n = 19), and the remaining were one 5′-UTR (CHEK2:c.-4C>T) and one splice variant (MLH1:c.678-4A>G).
All VUS variants identified in this study were reclassified by the Sherlock interpretation framework [17], and the results showed that all (100%) variants did not change from their previous VUS classification (Supplementary Table S2). Therefore, the clinical significance of the 21 VUSs described in this study remains unclear, since there is not sufficient evidence to associate them with a pathogenicity condition.

VUS, Variants Classification by ACMG Guidelines and Reclassification by SHERLOC Framework
In total, 24 VUS were found in 21 patients, since 2 of these patients (281/21 and 279/21) had more than one VUS. A detailed list of VUS variants identified in this study is provided in Table Table  S1). Considering a distinction for pathology, among the sixteen BC patients with no other LP/P variants, three harboured VUS in the BRCA1 gene (18.7%), four patients harboured VUS in the ATM (25%), five VUS in CHEK2 (31.2%), one in MSH6 gene (6.2%), one in MLH1 (6.2%) and two in PMS2 (12.5%) ( Table 2). As regards the germline variant type, most were missense variants (n = 19), and the remaining were one 5 -UTR (CHEK2:c.-4C>T) and one splice variant (MLH1:c.678-4A>G).
All VUS variants identified in this study were reclassified by the Sherlock interpretation framework [17], and the results showed that all (100%) variants did not change from their previous VUS classification (Supplementary Table S2). Therefore, the clinical significance of the 21 VUSs described in this study remains unclear, since there is not sufficient evidence to associate them with a pathogenicity condition.  Minor allele frequency (MAF), Clinical variation database [14]; Human Genome Variation Society [49]; nonreported (NR).

Likelihood of Carrying PVs in LP/P Variant-Positive HBOC and Prostate Cancer Patients versus Negative or Patients Carrying VUS
Considering the likelihood of carrying PVs in the moderate-to-high-risk genes in HBOC and prostate cancer patients, calculated by the BOADICEA model during genetic counselling with respect to the results of genetic testing, patients in the cohort were divided into three subgroups: (1) patients carrying VUS, (2) patients with LP/P pathogenic variants and (3) patients with no detected variants ( Figure 7A). Interestingly, the BOADICEA score was significantly higher in the likely pathogenic/pathogenic variant-positive HBOC and prostate cancer patients versus the variant-negative individuals or HBOC patients carrying the VUS variants ( Figure 7B). Although the study cohort was too limited, the ability of BOADICEA to predict BRCA1 and BRCA2 pathogenic variants seems better than the ability to predict pathogenic variants in other genes including PALB2, CHEK2, ATM, BARD1, RAD51D, RAD51C and BRIP1 (Supplementary Figures S1 and S2).

Discussion
The current study performed NGS analyses by evaluating 10 known disease-causing genes for HCPS on 104 enrolled patients from South Italy having a strong personal and family history chosen after genetic counselling. The major number of PVs were found in BRCA2 (n = 5), followed by BRCA1 (n = 4), TP53 (n = 3) and ATM (n = 2). PALB2, CHEK2 and MSH2 were found mutated in one patient only. Except for the PVs in ATM (c.9023G>A) found in the PC patient, TP53 (c.645del) in the patient affected by LFS, MSH2 (c.1204del) in the patient with colon cancer and BRCA1 (c.181C>T) in the OC patient, all the remaining PVs were found in the BC patients (n = 13).
The TP53 variant (c.645del) detected in patient number 764/19 was previously described in a Southern Italian family having an aggregated history of typical LFS cancers [11]. Germline TP53 PVs are associated with a wide range of cancers, known collectively as LFS, which is characterised by a predisposition towards a broad spectrum of malignancy, including soft-tissue sarcomas, adrenocortical carcinomas, brain tumours, early-onset BC and leukaemias [11].
Among the 13 BC-positive patients, 8/13 (61.5%) were in the BRCA1/2 genes. The PVs identified in BRCA1 were c.134+2T>G, c.134+2T>C and c.4964_4982del, whereas in BRCA2, they were c.8487+1G>A, c.7007G>A, c.1670T>G and c.1909+2T>A. The deletion c.4964_4982del in BRCA1, also known as 5083del19, was reported in the BC/OC patients by Baudi et al. as a founder mutation in South Italy (Calabria) [74] and was detected in several subjects with BC and OC [74,82]. The nonsense variant c.1670T>G, also known as L557X in the BRCA2, was reported in several subjects affected with BC or OC [39,83]. Interestingly, the BRCA2: c.1670T>G was found in two patients in this study cohort and a large study of the Italian population detected this PV in four Calabrian patients [84].
In addition to BRCA1/2, PALB2 is the most important gene involved in BC susceptibility [85]. Moreover, TP53, a well-known gene involved in Li-Fraumeni syndrome (LFS), is another high-penetrance BC susceptibility gene [86]. Two BC cases (488/21 and 22/22) that did not fulfil the classic LFS criteria were found to have PVs in TP53 (c.451C>G and c.376-1G>A). Our observations were consistent with that of a previous study in which some multiplex NGS panels for BC patients also detected several TP53 PVs that did not fulfil the classic LFS criteria [86]. Together with the previously described genes, ATM is also currently included in the major part of NGS panels for BC, since it is considered a moderate-penetrance BC susceptibility gene. In addition, it was observed that the relative risk of BC in a patient carrying a pathogenic variant of ATM is increased more than three times compared with the general population [21]. However, it has been observed that some variants in the ATM gene can be associated with a different degree of BC risk than other variants in the same gene. For example, the presence of the c.7271T>G PVs in ATM is associated with a significantly increased risk for early-onset BC, but the association between other PVs in ATM, such as 5557G>A or ivs38-8T>C, still remained unclear [87,88]. Therefore, although some germline PVs in the ATM gene have been shown to have an increased risk for BC, the role of ATM in BC risk is not fully understood, since the penetrance of some PVs appears similar to that of BRCA2 whereas others do not [88]. Although CHEK2 VPs are rare in BC, they can potentially contribute to BC susceptibility, since some interactions between mutated CHEK2 with other genes can be associated with BC development [29].
Interestingly, some PVs in CHEK2 seem to confer a higher cancer risk than others [89]. Currently, several methods and tools were developed in clinical genetic counselling to estimate the likelihood that a subject is a carrier of a PV in the BRCA1 or BRCA2 [90]. Among these models, BOADICEA can be used in clinical practice to promote genetic counselling and increase the prevention and surveillance of BC development [90]. Importantly, the BOADICEA model is accepted by the Care Excellence and NIH in the UK, which recommends a mutation probability threshold of 10% to select patients for genetic testing of BRCA1 and BRCA2 [90]. In this study, the likelihood of carrying PVs in the moderateto-high-risk genes in the HBOC and PC patients was evaluated by the BOADICEA model during genetic counselling. In the phase of genetic counselling, it should be assumed that the BOADICEA can estimate only the probability of carrying a PV in a subject and not the probability of detecting a PV [91]. To evaluate if the BOADICEA model can distinguish between carriers and noncarriers of PVs, the results of genetic tests were compared with the precalculated risks obtained for each patient during genetic counselling. Interestingly, this study shows that BOADICEA can distinguish between carriers and noncarriers of PVs, since the calculated score was significantly higher in the pathogenic variant-positive patients versus the variant-negative individuals with BC. However, we observed that some BC patients with high cancer risk BOADICEA scores resulted negatively for pathogenic variants (PVs). This could be because the detection of larger indels and exon-level copy number variants (CNVs) by NGS was not included in the workflow analysis of this study. In the current clinical practice, although only BRCA1 and BRCA2 are routinely evaluated in terms of large genomic rearrangements (LGRs), it has been observed that an important proportion of PALB2 PVs in BC subjects are LGRs [85]. Therefore, in addition to the BRCA1 and BRCA2 LRGs, PALB2 LGRs should also be included in routine clinical genetic testing.
The main limitations of this study are the limited size of the study cohort and the fact that the cohort is enriched for BC. In addition, this study did not include the detection of large rearrangements such as CNVs in BRCA1/2 or PALB2. Therefore, it cannot exclude that some patients of the study cohort at high risk of BC who were negative for single nucleotide variants (SNVs) and small insertions and deletions (Indels) are carriers of CNVs.
The purpose of this study in general is a description of the PVs found in the patients that were selected in our hospital by evaluating the extent and nature of PVs in the genes mainly implicated in HPCS. Another purpose of this study is to understand if our genetic counselling approach, combined with the multigene panel selected by us, has a clinical significance in hereditary cancer predisposition syndrome, especially in BC. Shortly, the trend is to use larger NGS panels for evaluating PVs in HPCS. However, although the addition of many moderate-to-low-risk genes into NGS panels could increase the diagnostic yield [92], this method can lead to complex findings, since the penetrance of particular germline variants of cancer-associated genes is yet to be defined.
The next step of this study will be the evaluation of CNVs in patients who were negative for SNV/indels by computational algorithms and their confirmation by orthogonal methods. In conclusion, this study shows that the multigene panel testing selected can offer an effective diagnostic approach for BC patients. In addition, the results of this study suggest that an accurate evaluation of the probability that the subject is a carrier of a germline PV in high-risk susceptibility genes is important to help counsellors to evaluate whether genetic testing is appropriate or not.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes13071286/s1, Table S1: Detailed information for each enrolled patient, including patient ID, age, sex, age of diagnosis, type of cancer, histological grade, molecular subtype, cancer onset, the occurrence of multiple tumours and the presence of affected first-and second-degree relatives, and BOADICEA scores; Table S2: Interpretation of VUS variants using ACMG rules and semiquantitative rules by Sherloc [46]; Figure S1: Likelihood of carrying PVs in BRCA1 and BRCA2 positive patients; Figure S2: Likelihood of carrying PVs in non-BRCA1/2 positive patients.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review of COMITATO ETICO SEZIONE AREA CENTRO, REGIONE CALABRIA (protocol n.113, 21 April 2022, Catanzaro).